IL-7/anti-IL-7 mAb complexes augment cytokine potency in mice through association with IgG-Fc and by competition with IL-7R

Abstract

Interleukin-7 (IL-7) is essential to T-cell survival as well as homeostatic proliferation, and clinical trials that exploit the mitogenic effects of IL-7 have achieved success in treating human diseases. In mice, the in vivo potency of IL-7 improves dramatically when it is administered as a complex with the anti-IL-7 neutralizing monoclonal antibody clone M25. However, the mechanism whereby M25 augments IL-7 potency is unknown. We have analyzed the discrete contributions of the antibody constant (Fc) and IL-7-binding (Fab) domains to the mechanism. By engaging the neonatal Fc receptor the Fc domain extends the in vivo lifespan of IL-7/M25 complexes and accounts for the majority of their activity. Unexpectedly, the IL-7-neutralizing Fab domain provides an additional, albeit smaller, contribution, possibly by serving as a cytokine depot. This study is the first to demonstrate that the neutralizing aspect of the monoclonal antibody is directly involved in enhancing the potency of a cytokine with a single form of receptor. Lessons from the mechanism of IL-7/M25 complexes inform the design of next-generation cytokine therapeutics.

Figures

Figure 1

Treatment with PTX and, to a lesser extent, FTY720 impairs IL-7/M25 potency in vivo. (A) Freshly isolated lymphocytes from B6.CD90.1+ donor mice were incubated in media alone or in media supplemented with 100 ng/mL PTX for 2 hours at 37°C, washed, labeled with CFSE, and injected intravenously (3 × 106 donor cells per host) into B6.CD90.2+ host mice on day 0. FTY720-treated hosts received 3 mg/kg FTY720 intraperitoneally on days −1 and 3. Host mice received intraperitoneal injections of either phosphate-buffered saline (PBS) or rhIL-7/M25 (1.5 μg/7.5 μg per injection) on days 1, 3, and 5. On day 7, host LNs and spleen (SPL) were harvested, and each host tissue was analyzed separately by flow cytometry. CFSE histograms of CD90.1+ CD8+ splenocytes (left) and total CD90.1+ CD8+ recovered from host LNs and SPL (right) are representative of at least 2 experiments with 1 to 2 hosts per condition. **P < .005; CRTL, control; ns, not significant. (B) Freshly isolated lymphocytes were treated with PTX or media alone (control) as described in (A) and cultured for 3 days at 37°C in complete RPMI media supplemented with the indicated concentrations of rhIL-7. Samples were stained with CD8-Pacific Blue and propidium iodide (PI), and the percentage of surviving cells (CD8+ PI−) was determined by flow cytometry. Each data point depicts one sample from an experiment. Results are representative of 3 experiments. (C) Frequency of donor cells recovered on day 7 from the LNs of individual hosts with or without PTX treatment, as described in (A); vertical bars indicate mean frequency for the cohort. Combined data from 3 similar experiments are shown; each point represents the frequency of CD90.1+ CD8+ lymphocytes for a single host. (D) CFSE-labeled B6.CD90.1+ lymphocytes (6 × 106) were injected into B6.CD90.2+ hosts treated as in (A) with rhIL-7/M25 and, where indicated, FTY720. On day 7, host spleen (SPL), inguinal (ILN), axillary (ALN), cervical (CLN), and mesenteric (MLN) lymph nodes were harvested separately, and CFSE dilution by CD90.1+ CD8+ cells was determined by flow cytometry. Results are representative of 2 experiments with 2 separately analyzed hosts per group.

Figure 2

IL-7/M25 requires FcRn, but not FcγR, to induce proliferation of CD8+ T cells in vivo. (A) On day 0, 8 × 106 CFSE-labeled CD8+ T cells purified from LNs of B6.CD90.1+ IL-7tg+ mice were adoptively transferred to the indicated B6.CD90.2+ hosts. Host mice received 3 intraperitoneal injections (inj.) of rhIL-7 and M25 (1.5 μg and 7.5 μg per injection) or PBS (data not shown) every other day. CFSE profiles of CD90.1+ CD8+CD44hi cells from day 7 host LNs are shown and are representative of 2 to 3 independent experiments with 2 separately analyzed mice per treatment. (B) CFSE-labeled CD8+ T cells (1.3 × 106) purified from B6.CD45.1+ LNs were adoptively transferred to B6.CD45.2+ hosts that were either wild-type (WT) or FcRn−/− (knockout [KO]). Host mice received intraperitoneal injections 3 times (days 1, 3, and 5) of either PBS or 1.5 µg rhIL-7 with or without 7.5 µg M25 or 6 injections (days 1 to 6) of 10 μg rhIL-7. Hosts were euthanized on day 7 and analyzed separately by flow cytometry. Shown here are CFSE histograms (LNs) and numbers of CD45.1+CD8+ T cells recovered from each host LN and spleen, with horizontal bars indicating the mean of each group. Data are representative of 2 experiments with 2 to 3 mice per treatment. *P < .05; **P < .005.

Figure 3

Multiple doses of IL-7 administered over 24 hours are unable to recapitulate the effect of a single dose of IL-7/M25. CD8+ T cells were purified from LNs of B6.CD90.1+ IL-7tg+ donor mice, CFSE-labeled, and injected intravenously (7 to 10 × 106 per host) into B6.CD90.2+ mice. (A) One day after adoptive transfer of donor cells, host mice received intraperitoneal injections of PBS, either a single injection of rhIL-7/M25 (1.5 μg/7.5 μg [middle left] or 10 μg/50 μg [middle right]), or a total of 12 doses of rhIL-7 alone (1.5 μg [bottom left] or 10 μg [bottom right]) administered every 2 hours for 24 hours. On day 7 after adoptive transfer, host LNs and spleen were harvested, and donor cell CFSE dilution was analyzed by flow cytometry. Shown are CFSE histograms of CD90.1+ CD8+ cells from host LNs that are representative of 2 to 3 experiments with 2 separately analyzed hosts per treatment. (B) Donor cells were allowed to park for 1 day following adoptive transfer, and then host mice received intraperitoneal injections of PBS, rhIL-7/M25 (3 μg/15 μg), or rhIL-7/M25Fab (3 μg/equivalent of 15 μg M25) once (third panel), or at 2-hour intervals over 24 hours (bottom). Six days after adoptive transfer, host LNs and spleens were harvested, and the CFSE histograms of CD90.1+ CD8+ donor cells were determined by flow cytometry. Histograms shown are representative of 2 experiments with 2 separately analyzed mice per condition. (C) C57BL/6 mice were injected intraperitoneally with PBS (ctrl, solid columns), 1.5 µg rhIL-7 (top panel), 1.5 µg /7.5 µg rhIL-7/M25 (middle panel), or 10 µg rhIL-7 (bottom panel) and euthanized at the indicated intervals posttreatment. Injections were staggered such that all mice in the same experiment could be analyzed at the same time. Mean fluorescent intensity (MFI) or relative geometric MFI (RFI) of CD127 on CD8+TCRβ+ [T-cell receptor β+] lymphocytes normalized to the average CD127 MFI of the PBS-treated mice from each experiment, which was set at 100% RFI, is shown from 3 combined experiments with 2 to 7 separately analyzed mice per condition (± standard error of the mean [SEM]). Significant difference from PBS-treated mice is indicated as *P < .05; **P < .005; ***P < .0005; ****P < .0001. hrs, hours; ND, not determined.

Figure 4

Association with an IgG Fc improves in vivo mitogenic effect of IL-7 on CD8+ T cells. (A) B6.CD45.2 hosts received intravenous injections of 4.5 × 106 CTV-labeled B6.CD45.1+ LN cells on day 0 and intraperitoneal injections on days 1, 3, and 5 of rhIL-7 (1.5 μg), rhIL-7/M25 (1.5 μg/7.5 μg), or IL-7-Fc (in vitro activity equal to 1.5 μg rhIL-7). Shown here are CTV histograms of CD45.1+ TCRβ+CD8+ cells from host LNs analyzed at day 7 representative of at least 3 experiments with 2 separately analyzed mice per treatment group. (B) CD8+ cells were purified from LN of B6.CD45.1+ donors, CFSE-labeled, and adoptively transferred (1.8 × 106 per host) by intravenous injection to CD45.2+ hosts, either wild-type or FcRn−/−, at day 0. Hosts received intraperitoneal injections on days 1, 3, and 5 of PBS alone, rhIL-7/M25 (3 μg/15 μg), or IL-7-Fc (equivalent of 3 μg rhIL-7). The number of CD45.1+ TCRβ+CD8+ cells recovered from host LNs and spleen at day 7 is shown (2 mice per group ± SEM). Results are representative of 2 experiments with 2 to 3 separately analyzed mice per group. *P < .05; **P < .005; ns, not significant.

Figure 5

M25Fab, but not M25, further improves the in vivo mitogenic effect of IL-7-Fc on CD8+ T cells. (A) B6.CD45.2+ host mice received 3.5 × 106 purified and CFSE-labeled CD45.1+CD8+ T cells intravenously on day 0 and intraperitoneal injections of PBS, IL-7-Fc (equivalent of 1.5 μg rhIL-7), or IL-7-Fc/M25 (equivalent of 1.5 μg rhIL-7/7.5 μg of M25) on days 2, 4, and 6. On day 8, donor cell proliferation in host LN and spleen was determined by flow cytometry. Shown are CFSE histograms of CD45.1+TCRβ+CD8+ lymphocytes representative of at least 3 experiments with 2 separately analyzed hosts per group. (B) CFSE-labeled B6.CD90.1+ LN cells (5 × 106) were adoptively transferred to B6.CD90.2+ hosts on day 0. Intraperitoneal injections administered to hosts on days 1, 3, and 5 consisted of PBS, rhIL-7/M25 (1.5 μg/7.5 μg), IL-7-Fc (equivalent of 1.5 μg rhIL-7), IL-7-Fc/M25 (equivalent of 1.5 μg rhIL-7/7.5 μg of M25), or IL-7-Fc/M25Fab (equivalent of 1.5 μg rhIL-7/equivalent of 7.5 μg M25). Day 7 CFSE profiles of CD90.1+TCRβ+CD8+ LN cells are depicted. Data are representative of at least 3 experiments with 1 to 4 separately analyzed mice per group. (C) Combined data from 2 identical experiments as described in (B), numbers of CD90.1+TCRβ+CD8+ cells recovered for LNs and spleens of mice treated with PBS (solid circles), IL-7-Fc (solid squares), or IL-7-Fc/M25Fab (solid triangles). Data are representative of 3 similar experiments. *P < .05. (D) B6.CD90.2+ hosts received 3 × 106 CFSE-labeled purified CD90.1+CD8+ T cells intravenously on day 0 and intraperitoneal injections on days 1, 3, and 5 of IL-7-Fc (IL-7 equivalents as indicated) alone or with M25Fab (M25 equivalents of either 3.75 μg, left; or 7.5 μg, right). Donor cell proliferation in host LNs and spleens was analyzed on day 7; shown are CFSE histograms gated on CD90.1+CD8+ lymphocytes that are representative of at least 2 experiments with 2 separately analyzed hosts per condition.